Metabolism Of Lysophospholipids Research Articles

Lipidomics reveals the dynamics of lipid profile altered by omega-3 polyunsaturated fatty acid supplementation in healthy people.

Glycerophospholipids (GPs) and sphingolipids (SPs) are important lipid components in the body and play biological functions. Omega-3 polyunsaturated fatty acids (n-3 PUFAs) are important nutrients, and their supplements are commonly used for preventing some diseases. However, the effect of n-3 PUFAs on the human glycerophospholipidome and sphingolipidome is unclear. We used targeted lipidomics to study the GP and SP profile of healthy individuals after supplementation with n-3 PUFAs for 3, 7, 14 and 21days. Fuzzy c-means clustering was used to cluster the lipid species into six classes reflecting different changed-content patterns after n-3 PUFA supplementation. Among the species with significantly changed content, lysophospholipids were the most sensitive; their content started to increase on day 3. The content of phosphatidylserines increased at a later stage. The content of most of the phosphatidylcholines and alkylphosphatidylcholines decreased on day 21. A correlation network analysis of lipid species suggested that some enzymes involved in the metabolism of lysophospholipids and phosphatidylserines were regulated by n-3 PUFAs. Levels of creatine kinase-MB (CK-MB), urea, glucose, triglycerides and total bilirubin were altered by n-3 PUFA at 21days. Correlation analysis revealed that the level of CK-MB was negatively correlated with those of species in lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine and phosphatidylserine classes, which were increased by n-3 PUFA supplementation. With the analysis in this work, we demonstrated the regular pattern of n-3 PUFAs on GP and SP metabolism, which provides a pharmacological basis for n-3 PUFAs for clinical application.

The membrane bound bacterial lipocalin Blc is a functional dimer with binding preference for lysophospholipids

Lipocalins, a widespread multifunctional family of small proteins (15–25 kDa) have been first described in eukaryotes and more recently in Gram-negative bacteria. Bacterial lipocalins belonging to class I are outer membrane lipoproteins, among which Blc from E. coli is the better studied. Blc is expressed under conditions of starvation and high osmolarity, conditions known to exert stress on the cell envelope. The structure of Blc that we have previously solved (V. Campanacci, D. Nurizzo, S. Spinelli, C. Valencia, M. Tegoni, C. Cambillau, FEBS Lett. 562 (2004) 183–188.) suggested its possible role in binding fatty acids or phospholipids. Both physiological and structural data on Blc, therefore, point to a role in storage or transport of lipids necessary for membrane maintenance. In order to further document this hypothesis for Blc function, we have performed binding studies using fluorescence quenching experiments. Our results indicate that dimeric Blc binds fatty acids and phospholipids in a micromolar K d range. The crystal structure of Blc with vaccenic acid, an unsaturated C18 fatty acid, reveals that the binding site spans across the Blc dimer, opposite to its membrane anchored face. An exposed unfilled pocket seemingly suited to bind a polar group attached to the fatty acid prompted us to investigate lyso-phospholipids, which were found to bind in a nanomolar K d range. We discuss these findings in terms of a potential role for Blc in the metabolism of lysophospholipids generated in the bacterial outer membrane.

Lysophosphatidic acid production and action: validated targets in cancer?

The completion of the human genome project, the evolution of transcriptional profiling and the emergence of proteomics have focused attention on these areas in the pathophysiology and therapy of cancer. The role of lysophospholipids as potential mediators in cancer pathophysiology, screening and management has taken a major leap forward with the recent cloning of several enzymes involved in the metabolism of lysophospholipids. Lysophospholipids, although small molecules, contain a high "informational" content. Differences include the nature of the phosphate head group, the regiochemistry of the fatty acyl chain on the glyceryl backbone, the presence of ether versus ester linkages to the backbone, and the length and saturation of the fatty acyl or alkyl chain. This informational content is sufficient to result in a marked structure function activity relationship at their cognate receptors. Thus the emerging discipline of "functional lipidomics" is likely to prove as important as genomics and proteomics in terms of early diagnosis, prognosis, and therapy. Lysophospholipid levels are elevated in vivo in a number of pathophysiological states including ascitic fluid from ovarian cancer patients indicating a role in the pathophysiology of this devastating disease. Although controversial, levels of specific lysophospholipids may be altered in the blood of cancer patients providing a potential mechanism for early diagnosis. Several of the enzymes involved in the metabolism of lysophospholipids are aberrant in ovarian and other cancers. Further, the enzymes are active in the interstitial space, rendering them readily accessible to the effects of inhibitors including antibodies, proteins, and small molecules. In support of a role for lysophospholipids in the pathophysiology of cancer, expression of receptors for lysophospholipids is also aberrant in cancer cells from multiple different lineages. All of the cell surface receptors for lysophospholipids belong to the G protein coupled receptor family. As over 40% of all drugs in current use target this family of receptors, lysophospholipid receptors are highly "druggable." Indeed, a number of highly specific agonists and antagonists of lysophospholipid receptors have been identified. A number are in preclinical evaluation as therapeutics. We look forward to the next several years when the role of lysophospholipids in physiology and the pathophysiology and management of cancer and other diseases are fully elucidated.

Unfolding the Pathophysiological Role of Bioactive Lysophospholipids

Lysophospholipids (LPLs), including glycerol- and sphingoid-based lipids, stimulate cell signaling and play important pathophysiological roles in humans and other animals. These LPLs include lysophosphatidic acid (LPA), lysophosphatidylinositol (LPI), lysophosphatidylcholine (LPC), lysophosphatidylserine (LPS), sphingosine-1-phosphate (S1P), and sphingosylphosphorylcholine (SPC). Analyses of LPLs in human body fluids from subjects with different pathophysiological conditions reveal not only the relevance of LPLs in human diseases, but also their potential application as biomarkers and/or therapeutic targets. In recent years, the identification and/or characterization of the plasma membrane receptors for LPLs and enzymes regulating the metabolism of LPLs have greatly facilitated our understanding of their role and signaling properties. In vitro and in vivo functional and signaling studies have revealed the broad and potent biological effects of LPLs and the mechanisms of LPL actions in different cellular systems. Development of specific antagonists for each of the LPL receptors will provide powerful tools for dissecting signaling pathways mediated by receptor subtypes. More importantly, these antagonists may serve as therapeutics for relevant diseases. Genetic depletion of LPL receptors in mice has provided and will continue to provide critical information on the pathophysiological roles of LPL receptors. It is important to further evaluate the significance of targeting these bioactive LPL receptors, their downstream signaling molecules, and/or metabolic enzymes in the treatment of cancers and other diseases.

Subcellular fractions of bovine brain degrade phosphatidylcholine by sequential deacylation of the sn-1 and sn-2 positions

Phosphatidylcholine (PC) metabolism was investigated using cytosol (fraction I) and particulate fractions of bovine brain that were enriched with microsomes (fraction II), plasma membranes (fraction III) or mitochondria (fraction IV). Fractions I–III incubated with 1- palmitoyl-2-[ 14 C]arachidonoyl-sn- glycero-3- phosphocholine yielded [ 14C]arachidonic acid at near equal rates, whereas only fraction I accumulated significant amounts of 2-[ 14 C]arachidonoyl-sn- glycero-3- phosphocholine . Much slower rates of arachidonic acid release were observed using an ether PC (1-O- hexadecyl-2-[ 3 H]arachidonoyl-sn- glycero-3- phosphocholine) . Moreover, arachidonic acid yield from the diacyl, but not ether PC was slowed by pretreating fractions I–III, but not IV, with phenylmethylsulfonyl fluoride (PMSF). Coincident with this decreased arachidonic acid, 2-[ 14 C]arachidonoyl-sn- glycero-3- phosphocholine was increased, indicating high PLA 1 activity. Taken together these data suggest that arachidonic release was largely dependent on initial deacylation of position sn-1. Incubating each untreated fraction with 2-[ 3 H]arachidonoyl-sn- glycero-3- phosphocholine yielded [ 3H]arachidonic acid (lysophospholipase A 2 activity) at rate that was substantially greater than that using the comparable PMSF-treated fraction. Thus, the large effect of PMSF on arachidonic acid release can be accounted for if much of the fatty acid formation arose from the sequential sn-1 and sn-2 deacylation of diacyl-PC by phospholipase A 1 and lysophospholipase A 2. When PMSF-treated fractions were incubated with 2-[ 3 H]arachidonoyl-sn- glycero-3- phosphocholine , [ 3H]PC accumulated at low rates that were enhanced by adding coenzyme A or stearoyl-coenzyme A. Thus, the lysophospholipid was also reacylated to form PC, but this reaction was negligible in the absence of PMSF and added cofactors. In summary, we conclude that, in brain subcellular fractions, deacylation of the sn-1 position of diacyl-PC proceeded more rapidly than sn-2 hydrolysis. There was substantial further metabolism of 2-acyl lysophospholipids due to the combined activities of a PMSF-sensitive and -insensitive lysophospholipase. Finally, the sequential deacylation of diacyl-PC by phospholipase A 1 and lysophospholipase A 2 probably accounted for the major portion of arachidonic acid produced.

Characterization of lysophospholipid metabolizing enzymes in human brain.

Lysophospholipids are generated during the turnover and breakdown of membrane phospholipids. We have identified and partially characterized three enzymes involved in the metabolism of lysophospholipids in human brain, namely, lysophospholipase, lysophospholipid:acyl-CoA acyltransferase (acyltransferase), and lysophospholipid:lysophospholipid transacylase (transacylase). Each enzyme displayed comparable levels of activity in biopsied and autopsied human brain, although in all cases the activity was somewhat lower in human than that in rat brain. All three enzymes were localized predominantly in the particulate fraction, with lysophospholipase possessing the greatest activity followed by acyltransferase and transacylase. Lysophosphatidylcholine possessed a Km in the micromolar range for lysophospholipase and transacylase, and in the millimolar range for acyltransferase, whereas arachidonyl-CoA displayed a Km in the micromolar range for acyltransferase. The three enzymes differed in their pH optima, with lysophospholipase being most active at pH 8.0, transacylase at pH 7.5, and acyltransferase at pH 6.0. Both bromophenacyl bromide and N-ethylmaleimide inhibited lysophospholipase activity and, to a lesser extent, that of acyltransferase and transacylase. None of the enzyme activities were affected by the presence of dithiothreitol or EDTA, although particulate lysophospholipase was activated approximately two-fold by the addition of 5 mM MgCl2 or CaCl2 but not KCl. Transacylating activity was stimulated by CoA, the EC50 of activation being 6.8 microM. Acyltransferase displayed an approximately threefold preference for arachidonyl-CoA over palmitoyl-CoA, whereas the acylation rate of different lysophospholipids was in the order lysophosphatidylinositol > 1-palmitoyl lysophosphatidylcholine > 1-oleoyl lysophosphatidylcholine >> lysophosphatidylserine > lysophosphatidylethanolamine. This, and the preference of human brain phospholipase A2 for phosphatidylinositol, suggests that this phospholipid may possess a higher turnover rate than the other phospholipid classes examined. Human brain homogenates also possessed the ability to transfer fatty acid from lysophosphatidylcholine to lysophosphatidylethanolamine. In addition, we also present evidence that diacylglycerophospholipids can act as acyl donors for the transacylation of lysophospholipids. We have therefore demonstrated the presence of, and partially characterized, three enzymes that are involved in the metabolism of lysophospholipids in human brain. Our results suggest that lysophospholipase may be the major route by which lysophospholipids are removed from the cell membrane in human brain. However, all three enzymes likely play an important role in the remodeling of membrane composition and thereby contribute to the overall functioning of membrane-associated processes.

Possible implication of lysophosphatidylcholine in cell fusion accompanying implantation in rabbits.

Implantation in rabbits involves the cellular fusion of trophoblastic and uterine epithelial cells resulting in embryo penetration of the uterine endometrium. Since lysophospholipids, known to have fusigenic properties, could be responsible for this cell fusion, the metabolism of lysophospholipids was studied throughout gestation in blastocyst/yolk sac and extracoelic amnioallantoic fluids. Analysis of phospholipid composition revealed that lysophospholipids are present in blastocyst/yolk sac fluid. Their concentrations and haemolytic activity change during pregnancy. They increase and reach their highest values during days 7 to 9, the implantation days in rabbits. A clear correlation was observed between lysophosphatidylcholine concentrations in blastocyst/yolk sac fluid and haemolysis induced by this fluid. Phosphatidylcholine concentrations, phospholipase A2 activity, which generates lysophospholipids, and lysophospholipase A activity which hydrolyses lysophosphatidylcholine into fatty acid, were at their highest value at day 12. These data suggest that a transient accumulation of lysophospholipids could ensure local cell fusion. Moreover, we propose that the lysophospholipid concentrations in blastocyst/yolk sac fluid are dependent upon activities of phospholipase A2 and lysophospholipase.

The acquisition of lipids by African trypanosomes.

Bloodstream forms of African trypanosomes are dependent on their host for fatty acids, choline and other components of membrane lipids. The bulk of their choline requirement is met by their ability to take up lysophospholipids from the host tissue fluids. Trypanosoma brucei has ocyltransferose and phospholipase A(1) activities for the metabolism of exogenous lysophospholipids. The rate of uptake of lysophospholipids can be controlled by changes in the extrocellulor concentration of fatty ocyl-coenzyme A, and this control has potential for chemotherapy.

Metabolism of phospholipids and lysophospholipids by Trypanosoma brucei

African trypanosomes ( Trypanosoma brucei brucei) rapidly metabolize exogenous 1-acyl-lysophospholipids by at least two routes: (1) hydrolysis by a phospholipase A1; (2) acylation by an acyl-CoA-dependent acyltransferase. In contrast to lysophospholipids, exogenous phospholipids are not rapidly metabolized by T. brucei. The acyltransferase (EC 2.3.1.23) converts exogenous 1-acyl lysophosphatidylcholine and exogenous acyl-CoA to phosphatidylcholine and CoA-SH. It is a membrane-bound enzyme and shows maximal activity within the first 2 min of exposure of trypanosomes to the exogenous substrates. The acyltransferase specificity for lysophospholipids is lysophosphatidylcholine > lysophosphatidylinositol > lysophosphatidylethanolamine > lysophosphatidate. Phosphatidylcholine enhances the enzyme activity towards lysophosphatidylethanolamine and lysophosphatidic acid. The preference for CoA acyl thioesters is oleoyl > palmitoyl > myristoyl > stearoyl > arachidonoyl, and this specificity distinguishes the protozoan enzyme from those of cells of mammalian hosts, which are specific for arachidonoyl-CoA. When the acyltransferase converts exogenous lysophosphatidylethanolamine to phosphatidylethanolamine, the latter is rapidly methylated to form dimethylphosphatidylethanolamine. There is also rapid hydrolysis of exogenous oleoyl-CoA by a thioester hydrolase in living trypanosomes, to yield free oleate and CoA-SH.

Metabolism of lysophospholipids in intact rat islets. The insulin secretagogue p-hydroxymercuribenzoic acid impairs lysophosphatidylcholine catabolism and permits its accumulation.

Although recent studies implicate lysophospholipids (lyso-PLs) in stimulus-secretion coupling in the pancreatic islet, almost no data on lyso-PL metabolism therein exist. Therefore, intact rat islets were loaded with insulinotropic and non-toxic concentrations of 1-[14C]palmitoyl-lysophosphatidylcholine (lyso-PC) via transbilayer movement, and its metabolic fate was studied. The time-dependent hydrolysis of lyso-PC to fatty acid (lysophospholipase activity), its conversion to phosphatidylcholine (putative acyltransferase activity) and, to a lesser degree, the appearance of label in phosphatidylethanolamine (putative transacylase or base exchange activity) were observed. p-Hydroxymercuribenzoic acid (PHMB) at 100 microM (a concentration previously demonstrated to elicit potent exocytotic insulin release) inhibited all three activities (by 56, 46 and 75%, respectively) and led to the intracellular accumulation of lyso-PC. Antimycin A inhibited phosphatidylcholine formation but not lysophospholipase activity; lyso-PC did not accumulate, implying that blockade of both of the major metabolic pathways is required to induce a detectable increment in lyso-PC levels. Calculations derived from data using the lowest effective insulinotropic concentration of lyso-PC suggested that increments in lyso-PC accumulation at critical membrane sites of less than 10-15% above basal values are sufficient to trigger insulin release. Since PHMB elicited increments of 50-100% in lyso-PC after its translocation into islets, support is provided for the earlier contention that lyso-PLs mediate the insulinotropic effect of PHMB. In addition, these studies may provide a more precise experimental paradigm for future studies of islet lyso-PL metabolism.

The effects of ischaemia, lysophosphatidylcholine and palmitoylcarnitine on rat heart phospholipase A2 activity.

Phospholipase A2 activity was studied in the isolated rat heart following coronary artery ligation. In both the homogenate and mitochondrial fractions phospholipase A2 activity was significantly depressed at 20 min post ligation in the ischaemic region only. This is at a time of peak lysophospholipid concentration and severity of arrhythmias. No such depression of activity was seen in a crude sarcolemmal fraction, possibly due to washout of inhibitory factors during isolation. Lysophosphatidylcholine and palmitoylcarnitine, two amphiphiles known to accumulate during ischaemia, were both shown to be capable of inhibiting phospholipase A2. It is suggested that lysophospholipid and palmitoylcarnitine accumulation during ischaemia may contribute to the depression of phospholipase A2 activity seen and that the decreased metabolism of lysophospholipids may be of more importance in their accumulation than increased production by phospholipase A2.

Incorporation and metabolism of 2-acyl lysophospholipids by Escherich1a coli

The incorporation of 2-acyl lysophospholipids into Escherichia coli, and their metabolism were studied. 2-[ 14C]Acyl lysophosphatidylethanolamine could penetrate into E. coli cells and was mainly incorporated into phosphatidylethanolamine. 2-Acyl lysophosphatidylethanolamine was partially degraded, but some of it was incorporated into membrane phospholipids by acylation. 2-Acyl lysophosphatidylcholine also entered cells and was acylated to phosphatidylcholine. The acylation of 2-acyl lysophospholipid by the envelope fraction was also studied. Fatty acids were incorporated into 2-acyl lysophospholipids by the envelope fraction in the presence of ATP and Mg 2+, and the incorporation was stimulated by acyl carrier protein, but not by coenzyme A. No acylation was observed with acyl coenzyme A as acyl donor. The acylation activities of the inner and outer membranes were examined. Pathways for degradation and modification of membrane phospholipids in E. coli are proposed.

Membrane lipid metabolism of bacillus calmette-guerin-induced rabbit alveolar macrophages

We examined the uptake of radiolabeled lysophospholipids and oleic acid by Bacillus Calmette-Guerin-induced rabbit alveolar macrophages either in the presence or absence of challenge particles. There was no difference in the uptake and metabolism of lysophospholipids by control or challenged cells for incubation periods up to 5 h. When incubated with [ 3H] oleic acid, challenged cells consistently exhibited a slightly greater uptake of radioactivity. Extraction of the whole cells revealed that the greater amount of radioactivity found in the challenged cells primarily was in triacylglycerol. There was no marked difference in the amount of radioactivity associated with the phospholipids in the whole cell extracts from control and challenged cells. When the macrophages were pre-labeled for 15 min with [ 3H]oleic acid and then reincubated in fresh medium in the presence or absence of autoclaved Escherichia coli B, more radioactivity was retained by the challenged cells, again in the form of triacylglycerol. Only in isolated plasma membrane fractions did we observe a difference in the amount of radioactivity associated with phospholipids from control and challenged cells. Plasma membranes isolated from Bacillus Calmette-Guerin-induced rabbit alveolar macrophages that had been incubated for 6 h with [ 3H] oleic acid in the presence of E. coli B contained a significantly higher level of radioactivity in all lipids than plasma membranes from control cells. Since the greatest and the most consistent difference between control and challenged cells is associated with the triacylglycerol molecule, it is postulated that this molecule may serve as a precursor in the synthesis of alveolar macro-phage phospholipids, both by the reacylation pathway and the de novo pathway. It is possible that the high level of radiolabeled phospholipid found in the plasma membrane arose via the de novo pathway following the cleavage of an acyl group as we have found cytidine diphosphocholine phosphotransferase in the plasma membrane fraction (Wang, P., DeChatelet, L.R.and Waite, M. (1977) Biochim. Biophys. Acta 450, 311–321).